Cole E S, Lepp C A, Holohan P D, Fondy T P
J Biol Chem. 1978 Nov 10;253(21):7952-9.
Mitochondrial glycerol-3-P dehydrogenase (EC 1.1.99.5) has been purified in 20% yield from both rabbit skeletal muscle and brain using a four step procedure involving osmotic shock, solubilization with Triton X-100, hydrophobic chromatography, gel filtration, and preparative column isoelectrofocusing. The active muscle and brain enzymes were found to be 95% and 80% homogeneous, respectively. Final purification was performed on the denatured subunit. The active enzyme from each of the tissues focused at pH 5.25 +/- 0.12 and each produced similar biphasic thermal inactivation plots at 50 degrees C. Mixtures of the purified brain and muscle enzymes co-migrated in discontinuous electrophoresis gels and each enzyme exhibited a single polypeptide component on sodium dodecyl sulfate (SDS) gels either when run separately or in mixtures. The subunit molecular weight was shown to be 76,000 +/- 3,000 by SDS-gel electrophoresis and gel filtration in 6 M guanidine HCl. One mole of noncovalently bound FAD and 1 mole of iron were measured per Mr = 100,000. The amino acid composition was determined based on the assumption of 70 aspartate residues per subunit to give a Mr = 76,000. The absorption spectrum has a maximum at 416 nm and a shoulder at 450 to 460 nm which is bleached on treatment with sodium dithionite. The maximum at 416 nm is removed by treatment with mersalyl.
已采用包括渗透休克、用Triton X - 100增溶、疏水色谱、凝胶过滤和制备性柱等电聚焦在内的四步程序，从兔骨骼肌和脑中以20%的产率纯化了线粒体甘油-3-磷酸脱氢酶（EC 1.1.99.5）。发现活性肌肉酶和脑酶分别具有95%和80%的纯度。最终纯化是在变性亚基上进行的。来自每个组织的活性酶在pH 5.25±0.12处聚焦，并且在50℃时每种酶都产生相似的双相热失活曲线。纯化的脑酶和肌肉酶的混合物在不连续电泳凝胶中共迁移，并且每种酶在十二烷基硫酸钠（SDS）凝胶上单独运行或混合运行时均显示出单一多肽组分。通过SDS - 凝胶电泳和在6 M盐酸胍中的凝胶过滤，亚基分子量显示为76,000±3,000。每Mr = 100,000测量到1摩尔非共价结合的FAD和1摩尔铁。基于每个亚基70个天冬氨酸残基的假设确定氨基酸组成，以给出Mr = 76,000。吸收光谱在416 nm处有最大值，在450至460 nm处有一个肩峰，在用连二亚硫酸钠处理时会褪色。416 nm处的最大值在用汞撒利处理时会消失。
